Circadian rhythms, found in most eukaryotes and some prokaryotes (Kay et al, Cell, 83:361 (1995)), are .about.24 hour rhythms governed by an internal clock that functions autonomously, but can be entrained by environmental cycles of light or temperature. Circadian rhythms produced in constant darkness can also be reset by pulses of light. Such light pulses will shift the phase of the clock in different directions (advance or delay) and to varying degrees in a fashion that depends on the time of light exposure (Pittendrigh, in Handbook of Behavioral Neurobiology, 4, J. Aschoff, Ed., New York: Plenum, 1981, pp. 95-124).
Fruit flies show circadian regulation of several behaviors (Pittendrigh in The Neurosciences Third Study Program, F. O. Schmitt and F. G. Worden, Eds. (MIT Press, Cambridge Mass., 1974, Chap. 38; Jackson, in Molecular Genetics of Biological Rhythms, M. W. Young, Ed. (Dekker, New York, 1993), pp. 91-121). When populations of Drosophila are entrained to 12 hours of light followed by 12 hours of darkness (LD 12:12), adults emerge from pupae (enclose) rhythmically, with peak eclosion recurring every morning. The eclosion rhythm persists when the entraining cues are removed and behavior is monitored in constant darkness, thus indicting the existence of an endogenous clock. Adult locomotor activity is also controlled by an endogenous clock and recurs rhythmically with a 24-hour period.
Mutations in the Drosophila period (per) gene disrupt circadian rhythms of pupal eclosion and adult locomotor behavior (Konopka and Benzer Proc. Natl. Acad. Sci. U.S.A. 68:2112 (1971)). Although per has been cloned and sequenced and its pattern of expression has been analyzed (Baylies et al in Molecular Genetics of Biological Rhythms, M. W. Young, Ed. (Dekker, New York, 1993), pp. 123-153; Rosbash and Hall Neuron 3:387 (1989)), the biochemical function of the PER protein is unknown. PER shares some homology with a family of transcription factors (Crews et al Cell 52:143 (1988); Nambu et al Cell 67:1157 (1991); Reisz-Porszasz et al Science 256:1193 (1992); Hoffman et al Cell 252:954 (1991); Burbach et al Proc. Natl. Acad. Sci. U.S.A. 89:8185 (1992)) that possess a common sequence motif called the PAS domain. The PAS domain consists of two repeats of approximately 50 amino acids within a homology region of 258 to 308 amino acids. Sequence similarity between PER and protein regulating aspects of Drosophila development, SIM (encoded by the gene single-minded), was first recognized by Crews et al (Jackson et al J. Neurogenet 1:3 (1983); Dushay et al J. Biol. Rhythms 4:1 (1989); Dushay et al Genetics 125:557 (1990); Konopka et al J. Neurogenet 7:103 (1991)). Subsequent studies have shown that this homology region is also found in ARNT (arylhydrocarbon receptor nuclear translocator), a component of the multisubunit mammalian dioxin receptor (Reyes et al Science 256:1193 (1992); Hoffman et al Neuron 252:954 (1991)), and in AHR, the ligand-binding subunit of the dioxin receptor (Burbach et al Proc. Natl. Acad. Sci. U.S.A 89:8185 (1992)). The region of homology is now referred to as the PAS domain, after the first three proteins in which it was identified: PER, ARNT, and SIM (Huang et al Nature 364:259 (1993)).
Immunocytochemical experiments demonstrated that PER is a nuclear protein in a variety of Drosophila tissues (Konopka and Benzer (1971); Baylies et al (1993)). In cells of the adult fly visual and nervous systems, the amount of PER protein fluctuates with a circadian rhythm (Edery et al Proc. Natl. Acad. Sci. U.S.A 91:2260 (1994)), the protein is phosphorylated with a circadian rhythm (Edery et al (1994)), and PER is observed in nuclei at night but not late in the day (Siwicki et al Neuron 1:141 (1988); Saez and Young Mol. Cell. Biol. 8:5378 (1988); Zerr et al J. Neurosci 10:2749 (1990)). The expression of per RNA is also cyclic. However, peak mRNA amounts are present late in the day, and the smallest amounts are present late at night (Konopka and Benzer (1971)). Three mutant alleles--per.sup.O, per.sup.S, and per.sup.L,--cause arrhythmic behavior or shorten or lengthen periods, respectively (Konopka and Benzer (1971)). These mutations also produce corresponding changes in the rhythms of per RNA and protein amounts (Edery et al (1994); Hardin et al Nature 343:536 (1990); Proc. Natl. Acad. Sci. U.S.A. 89:11711 (1992); Sehgal et al Science 263:1603 (1994)) and PER immunoreactivity in nuclei (Sewicki et al Neuron 1:141 (1988); Saez and Young Mol. Cell. Biol. 8:5378 (1988); Zerr et al J. Neurosci. 10:2749 (1990)). This suggests a possible role for molecular oscillations of per in the establishment of behavioral rhythms (Hardin et al (1992)). Several mutations that affect eclosion and locomotor activity have been isolated in behavioral screens (Jackson (1993); Konopka and Benzer (1971); Rosbash and Hall (1989); Baylies et al (1993) Jackson (1983); Dushay et al (1989); Dushay et al (1990); Konopka et al (1991)). The best characterized, and those with the strongest phenotypes, are mutations at the X chromosome-linked period (per) locus (Konopka and Benzer (1971); Rosbash and Hall (1989); Baylies et al (1993) Jackson (1983); Dushay et al (1989); Dushay et al (1990)). Missense mutations at per can lengthen or shorten the period of circadian rhythms, whereas null mutations abolish circadian rhythms altogether. The per gene is expressed in many cell types at various stages of development. In most cell types, the period protein (PER) is found in nuclei (James et al EMBO J. 5:2313 (1986); Liu et al Genes Dev. 2:228 (1988); Saez and Young Mol. Cell. Biol. 8, 5378 (1988); Liu et al J. Neurosci. 12:2735 (1992) Siwicki et al Neuron 1:141 (1988); Zerr et al J. Neurosci. 10:2749 (1990); Edery et al Proc. Natl. Acad. Sci. U.S.A. 91:2260 (1994)). A domain within PER is also found in the Drosophila single-minded protein (SIM) and in subunits of the mammalian aryl hydrocarbon receptor (Crews et al Cell 52:143 (1988); Hoffman et al Science 252:954 (1991); Burbach et al Proc. Natl. Acad. Sci. U.S.A. 89:8185 (1992); Reyes et al Science 256:1193 (1992)), and this domain (PAS, for PER, ARNT, and SIM) mediates dimerization of PER (Huang et al Nature 364:259 (1993)). The amounts of both PER protein and RNA oscillate with a circadian period, which is affected by the per mutations in the same manner as behavioral rhythms are affected (Siwicki et al (1988); Zerr et al (1990); Edery et al (1994); Hardin et al Nature 343:536 (1990); Proc. Natl. Acad. Sci. U.S.A. 89:11711 (1992)). Given the homologies to sim and the aryl hydrocarbon receptor (which are thought to regulate transcription), the effects of per on behavioral rhythms have been postulated to depend on circadian regulation of gene expression, including that of per itself (Hardin et al Nature 343:536 (1990); Proc. Natl. Acad. Sci. U.S.A. 89:11711 (1992)). However, neither direct proof of this postulate nor elucidation of per's actual biochemical function has been forthcoming.